Biological Engineering Divisionhttp://hdl.handle.net/1721.1/7752
Thu, 12 Oct 2017 11:13:47 GMT2017-10-12T11:13:47ZExpansion microscopy : scalable and multiplexed nanoscale imaginghttp://hdl.handle.net/1721.1/111502
Expansion microscopy : scalable and multiplexed nanoscale imaging
Chen, Fei, Ph. D. Massachusetts Institute of Technology. Department of Biological Engineering
Microscopy has facilitated the discovery of many biological insights by optically magnifying small structures in cells and tissues. However, the resolution of optical microscopy is limited by the diffraction of light to ~200-300 nm, comparable or larger to the size of many subcellular structures. In this thesis, we describe a suite of tools based on a novel super-resolution microscopy approach called Expansion microscopy. Expansion microscopy (ExM) physically expands tissues so that the resolution of ordinary microscopes is increased -5 times by leveraging the swelling properties of polyelectrolyte hydrogels. Ordinary microscopes used with ExM are more accessible and faster than the specialized optical systems designed to image beyond the diffraction limit (e.g., STORM/PALM, STED, SIM), while yielding similar performance. Expanded tissues are also optically clear, allowing for unprecedented super-resolution imaging in thick tissues and facile reagent diffusion into the sample. We have since developed a variant of ExM, called protein retention ExM, in which proteins are directly anchored to the swellable gel using a commercially available cross-linking molecule. This strategy enables ExM of genetically encoded fluorescent proteins and commercial fluorescently labeled secondary antibodies. With these advancements, ExM can be carried out with purely commercial reagents and represents a simple extension of standard histological methods used to prepare samples for imaging. Furthermore, we have developed a variant of the ExM technology that enables RNA molecules to be directly linked to the ExM gel network via a small molecule linker and isotropic expansion. This technology, termed ExFISH, enables visualization of RNAs with nanoscale precision and single molecule resolution. We have demonstrated that the covalent anchoring of RNA also enables robust repeated washing and probe hybridization steps, opening the door to combinatorial multiplexing strategies. By leveraging these benefits, we have further developed in situ analysis tools which allow for highly multiplexed imaging of RNA identity and location with nanoscale precision in intact tissues. Taken together, these tools allow for spatially mapping molecular information onto cell types and tissue structures which could be invaluable for spatially complex biological processes such as brain function, cancer heterogeneity and organismal development.
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017.; Cataloged from PDF version of thesis.; Includes bibliographical references (pages 99-107).
Sun, 01 Jan 2017 00:00:00 GMThttp://hdl.handle.net/1721.1/1115022017-01-01T00:00:00ZSystems biology of diet-induced hepatic insulin resistancehttp://hdl.handle.net/1721.1/111272
Systems biology of diet-induced hepatic insulin resistance
Soltis, Anthony Robert
Human obesity is a world-wide health crisis that promotes insulin resistance and type 2 diabetes. Obesity increases intracellular free fatty acid concentrations in peripheral tissues, particularly the liver, which disrupts molecular mechanisms that maintain normal glycemia in response to fasting and feeding. The progression towards outright pathology in response to obesity is a highly complex process that involves coordinated dysregulation of a variety of molecular processes across multiple regulatory levels. The goal of this thesis was to apply a quantitative, multi-omic systems biology approach to the study of obesity-induce hepatic insulin resistance. We fed male C57BL/6J mice high-fat diets (HFD) to induce obesity and insulin resistance. In the first presented study, our group collected datasets to profile the hepatic epigenomes, transcriptomes, proteomes, and metabolomes of chow diet (CD) control and HFD-fed mice. I extended and applied an established computational modeling algorithm, namely the prize-collecting Steiner forest (PCSF), to simultaneously integrate these molecular data with protein-protein and protein-metabolite interactions into a tractable network model of hepatic dysregulation. This model uncovered a variety of dysregulated pathways and processes, some of which are not well-established aspects of insulin resistance. We further tested and validated some of these model predictions, finding that HFD induces serious architectural defects in the liver and enhances hepatocyte apoptosis. In the next study, we focused more specifically on hepatic transcription. We fed mice short and long-term HFDs and treated them with the type 2 diabetes drug metformin. Compared to non-treated CD controls, diet exerted the strongest effect on transcription, progressively inducing changes as HFD duration increased. We additionally stimulated mice with insulin and collected temporal transcriptomic profiles. We found that long-term HFD almost completely blunted normal insulin-induced transcriptional changes, but also found a small set of genes that are specifically insulin-responsive in HFD livers. We further characterized one of these genes and provided evidence supporting the notion that aspects of hepatic insulin signaling are intact during insulin resistance. In another study, we collected transcriptomic and epigenomic data from mice fed a calorie-restricted (CR) diet. Interestingly, we found a small set of genes altered in the same direction by both CR and HFD. We then used chromatin accessibility experiments to infer regulators associated with these gene expression changes and found roles for PPAR[alpha] and RXR[alpha]. We performed ChIP-Seq experiments for these factors and treated mice and primary hepatocytes with a PPAR[alpha] activator, uncovering a role for PPARα in the regulation of anaerobic glycolysis. We also validated novel predicted target genes of PPAR[alpha] involved in glucose metabolism. Finally, we profiled hepatic miRNAs in CD and HFD livers, finding that HFD progressively alters their expression levels. We implemented an enrichment procedure and a network modeling approach to analyze these data. We integrated additional mRNA and epigenetic data to infer miRNAs that may play regulatory roles during insulin resistance. In total, this thesis presents a unique comprehensive approach to the study of diet-induced hepatic insulin resistance that revealed new insights into pathology.
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017.; This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.; Cataloged from student-submitted PDF version of thesis.; Includes bibliographical references (pages 192-205).
Sun, 01 Jan 2017 00:00:00 GMThttp://hdl.handle.net/1721.1/1112722017-01-01T00:00:00ZDecoding structure-function relationships of glycanshttp://hdl.handle.net/1721.1/110887
Decoding structure-function relationships of glycans
Stebbins, Nathan Wilson
Glycans are an important class of biological molecules that regulate a variety of physiological processes such as signal transduction, tissue development and microbial pathogenesis. However, due to the structural complexity of glycans and the unique intricacies of glycan-protein interactions, elucidating glycan structure-function relationships is challenging. Thus, uncovering the biological function of glycans requires an integrated approach, incorporating structural analysis of glycans, and glycan-proteins interactions with functional analysis. In this thesis, I develop new tools and implement integrated approaches to study glycans and glycan-binding proteins (GBPs). I apply these approaches to study glycans and GBPs in two areas: i) the role of hemagglutinin-glycan receptor specificity in human adaptation and pathogenesis of influenza and ii) the function of glycan regulation of cell-microenvironment interaction in cancer progression. Section 1: Influenza poses a significant public health threat and there is a constant looming threat of a pandemic. Pandemic viruses emerge when avian viruses acquire mutations that enable human adaptation, leading to infection of an antigenically naive host. Influenza Hemagglutinin (HA), and HA-glycan receptor interactions, play a central role in host tropism, transmissibility, and immune recognition. In section one, I develop and apply an integrated approach comprised of structural modeling, inter-amino acid network analysis, biochemical assays, and bioinformatics tools to study the hemagglutinin-glycan interaction and, in some cases, HA's antigenic properties. Using this approach, we i) identify the structural determinants required, and potential mutational paths, for H5N1 to quantitatively switch it's binding specificity to human glycans receptors, ii) identify the mutations that enable the 2013 outbreak H7N9 HA to improve binding to human glycan receptors in the upper respiratory tract, iii) uncover H3N2 strains that are currently circulating in birds and swine that possess features of a virus that could potentially re-emerge and cause a pandemic, and iv) characterize the glycan binding specificity of a novel 2011 Seal H3N8 HA. The approaches implemented here and the findings of these studies provide a framework for improved surveillance of influenza viruses circulating in non-human hosts that pose a pandemic threat. Section 2: Glycans are abundant on the cell surface, and at the cell-ECM interface where they mediate interactions between cells and their microenvironment. Despite this, the function of glycans in cancer progression remains largely understudied. Here, I develop an integrated approach to characterize the cell surface glycome, including N-linked, 0-linked glycans, and HSGAGs. This approach integrates glycogene expression data, analytical tools, and glycan binding protein reagents. I demonstrate that this platform enables rapid and efficient characterization of the N- and 0-linked glycome in a model cell system, representing metastatic versus non-metastatic cancer cells. Next, I apply this integrated approach to uncover new roles of glycans. I study the role that HSGAGs play in regulating cancer stem cell (CSC) activity in breast cancer. Here, we report that SULF1, an HSGAG modifying enzyme, is required for efficient tumor initiation, growth and metastasis of CSCs. Furthermore, we identify a putative mechanism by which SULF1 regulates interactions between CSCs and their microenvironment. The approaches implemented here and the finding of these studies Overall, this thesis provides important tools, approaches and insights to enable and improve the study of glycans and glycan binding proteins. Together the work here provides a framework for decoding structure-function relationship of glycans.
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017.; Cataloged from PDF version of thesis.; Includes bibliographical references (pages 236-275).
Sun, 01 Jan 2017 00:00:00 GMThttp://hdl.handle.net/1721.1/1108872017-01-01T00:00:00ZMolecular pathway analysis and therapeutics development in post-traumatic osteoarthritishttp://hdl.handle.net/1721.1/109668
Molecular pathway analysis and therapeutics development in post-traumatic osteoarthritis
Wang, Yang, Ph. D. Massachusetts Institute of Technology
Post traumatic osteoarthritis (PTOA) refers to the progressive degradation of cartilage often triggered by a traumatic joint injury, such as a tear of the meniscus or anterior cruciate ligament (ACL). Such impact injuries lead to elevated levels of inflammatory cytokines in the synovial fluid of the joint, including IL-1, IL-6, and TNFa. In turn, these cytokines cause decreased matrix synthesis by chondrocytes and contribute to reprogramming of chondrocytes and synovial cells to increase release of matrix proteases. PTOA accounts for 12% of the OA population and typically affects younger individuals. The first part of this thesis focuses on developing a combination therapeutic which can address multiple aspects of cartilage degradation associated with the pathogenic responses to joint injury. We studied the combined use of insulin-like growth factor 1 (IGF-1) and dexamethasone (Dex) to block multiple degradative effects of cytokine challenge to articular cartilage. We found that in young bovine cartilage, the combination of IGF- 1 and Dex significantly inhibited the loss of sulfated glycosaminoglycans (sGAG) and collagen induced by IL-I. rescued the suppressed matrix biosynthesis, and inhibited the loss of chondrocyte viability caused by iL- 1 treatment. In adult human cartilage, only IGF- 1 rescued matrix biosynthesis and only Dex inhibited sGAG loss and improved cell viability. Thus, the combination of IGF-1+Dex together showed combined beneficial effects in human cartilage. Our findings suggest that the combination of IGF-I and Dex has greater beneficial effects than either molecule alone in preventing cytokine-mediated cartilage degradation in adult human and young bovine cartilage. In the second part of this thesis, a global phosphoproteomics approach was employed to determine the pathways that are activated upon cytokine challenge of adult human chondrocytes. We identified key regulatory kinases, p38, JNKI/2, ERKI/2, ERK5, JAK2, and STAT3 that were upregulated in phosphorylation as a result of inflammatory cytokine treatment. In addition, we identified 417 phosphopeptides with MAPK substrate motif that were more than 4 times upregulated in response to cytokine treatment. Using inhibitors against the key kinases, it was shown that P38, JNK1/2, ERK5 played important roles in cytokine induced cell death in bovine and human cartilage, while inhibition of JNK1/2 and ERK5 had the anti-catabolic effect of reducing GAG loss from cartilage matrix. In addition, JNK inhibition sensitized chondrocytes to IGF-1 stimulation in young bovine cartilage. These result indicate that kinase activity plays an essential role in cytokine induced cartilage catabolism and that kinase inhibitors have therapeutic potential in preventing cartilage degeneration. The third and final part of this work examined the release of matrix molecules upon mechanical injurious compression and/or cytokine treatment in long term culture to identify potential biomarkers of cartilage degeneration. A quantitative mass spectrometry approach was used to characterize the kinetics of aggrecan and collagen degradation. Although mechanical injury alone does not lead to a substantial increase in matrix degradation, mechanical injury can accelerate cytokine-induced matrix degradation and release. Additionally, we found that a collagen type III neo-epitope could be a potential biomarker for cartilage degradation. A neoepitope of cartilage oligomeric matrix protein (COMP), which was identified in the synovial fluid of acute injury patients, was also found in our ex vivo explant injury model. This makes our model physiologically relevant and it can be a valuable system for determining the effects of potential drug treatment on matrix degradation.
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, February 2017.; Cataloged from PDF version of thesis.; Includes bibliographical references.
Sun, 01 Jan 2017 00:00:00 GMThttp://hdl.handle.net/1721.1/1096682017-01-01T00:00:00Z